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The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.

Human-induced pluripotent stem cells (hiPSCs) offer a promising source for generating dental epithelial (DE) cells. Whereas the existing differentiation protocols were time-consuming and relied heavily on growth factors, herein, we developed a three-step protocol to convert hiPSCs into DE cells in 8 days. In the first phase, hiPSCs were differentiated into non-neural ectoderm using SU5402 (an FGF signaling inhibitor). The second phase involved differentiating non-neural ectoderm into pan-placodal ectoderm and simultaneously inducing the formation of oral ectoderm (OE) using LDN193189 (a BMP signaling inhibitor) and purmorphamine (a SHH signaling activator). In the final phase, OE cells were differentiated into DE through the application of Purmorphamine, XAV939 (a WNT signaling inhibitor), and BMP4. qRT-PCR and immunostaining were performed to examine the expression of lineage-specific markers. ARS staining was performed to evaluate the formation of the mineralization nodule. The expression of PITX2, SP6, and AMBN, the emergence of mineralization nodules, and the enhanced expression of AMBN and AMELX in spheroid culture implied the generation of DE cells. This study delineates the developmental signaling pathways and uses small molecules to streamline the induction of hiPSCs into DE cells. Our findings present a simplified and quicker method for generating DE cells, contributing valuable insights for dental regeneration and dental disease research.

Induced pluripotent stem cells (iPSCs) may develop into any form of cell and are being intensively investigated. The influence on iPSCs of nanostructures generated using two-dimensional colloidal arrays was examined in this study. Colloidal arrays were formed using the following procedure. First, core–shell colloids were adsorbed onto a glass substrate using a layer-by-layer method. Second, the colloids were immobilized via thermal fusion. Third, the surface of the colloids was modified by plasma treatment. By adjusting the number density of colloids, cultured iPSCs were easily detached from the substrate without manual cell scraping. In addition to planar culture, cell aggregation of iPSCs attached to the substrate was achieved by combining hydrophilic surface patterning on the colloidal array. Multilayered cell aggregates with approximately four layers were able be cultured. These findings imply that colloidal arrays might be an effective tool for controlling the strength of cell adhesion.

Mitochondria are critical organelles responsible for the maintenance of cellular energy homeostasis. Thus, their dysfunction can have severe consequences in cells responsible for energy-intensive metabolic function, such as hepatocytes. Extensive research over the last decades have identified compromised mitochondrial function as a central feature in the pathophysiology of liver injury induced by an acetaminophen (APAP) overdose, the most common cause of acute liver failure in the United States. While hepatocyte mitochondrial oxidative and nitrosative stress coupled with induction of the mitochondrial permeability transition are well recognized after an APAP overdose, recent studies have revealed additional details about the organelle’s role in APAP pathophysiology. This concise review highlights these new advances, which establish the central role of the mitochondria in APAP pathophysiology, and places them in the context of earlier information in the literature. Adaptive alterations in mitochondrial morphology as well as the role of cellular iron in mitochondrial dysfunction and the organelle’s importance in liver recovery after APAP-induced injury will be discussed.

Oxygen consumption has been used to evaluate various cellular activities. In addition, three-dimensional (3D) spheroids have been broadly exploited as advanced in vitro cell models for various biomedical studies due to their capability of mimicking 3D in vivo microenvironments and cell arrangements. However, monitoring the oxygen consumption of live 3D spheroids poses challenges because existing invasive methods cause structural and cell damage. In contrast, optical methods using fluorescence labeling and microscopy are non-invasive, but they suffer from technical limitations like high cost, tedious procedures, and poor signal-to-noise ratios. To address these challenges, we developed a microfluidic platform for uniform-sized spheroid formation, handling, and culture. The platform is further integrated with widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) to efficiently characterize the lifetime of an oxygen-sensitive dye filling the platform for oxygen consumption characterization. In the experiments, osteosarcoma (MG-63) cells are exploited as the spheroid model and for the oxygen consumption analysis. The results demonstrate the functionality of the developed approach and show the accurate characterization of the oxygen consumption of the spheroids in response to drug treatments. The developed approach possesses great potential to advance spheroid metabolism studies with single-spheroid resolution and high sensitivity.

The expression of pluripotency factors, and their associations with clinicopathological parameters and drug response have been described in various cancers, including gastric cancer. This study investigated the association of pluripotency factor expression with the clinicopathological characteristics of gastric cancer patients, as well as changes in the expression of these factors upon the stem cell-enriching spheroid culture of gastric cancer cells, regulation of sphere-forming capacity, and response to cisplatin and TRAIL treatments by Nanog and KLF4. Nanog expression was significantly associated with the emergence of a new tumor and a worse prognosis in gastric cancer patients. The expression of the pluripotency factors varied among six gastric cancer cells. KLF4 and Nanog were expressed high in SNU-601, whereas SOX2 was expressed high in SNU-484. The expression of KLF4 and SOX2 was increased upon the spheroid culture of SNU-601 (KLF4/Nanog-high) and SNU-638 (KLF4/Nanog-low). The spheroid culture of them enhanced TRAIL-induced viability reduction, which was accompanied by the upregulation of death receptors, DR4 and DR5. Knockdown and overexpression of Nanog in SNU-601 and SNU-638, respectively, did not affect spheroid-forming capacity, however, its expression was inversely correlated with DR4/DR5 expression and TRAIL sensitivity. In contrast, KLF4 overexpression in SNU-638 increased spheroid formation, susceptibility to cisplatin and TRAIL treatments, and DR4/DR5 expression, while the opposite was found in KLF4-silenced SNU-601. KLF4 is supposed to play a critical role in DR4/DR5 expression and responses to TRAIL and cisplatin, whereas Nanog is only implicated in the former events only. Direct regulation of death receptor expression and TRAIL response by KLF4 and Nanog have not been well documented previously, and the regulatory mechanism behind the process remains to be elucidated.

Managing metastasis at the early stage and detecting and treating submillimeter tumors at early metastasis are crucial for improving cancer prognosis. Angiogenesis is a critical target for developing drugs to detect and inhibit submillimeter tumor growth; however, drug development remains challenging because there are no suitable models for observing the submillimeter tumor mass and the surrounding blood vessels in vivo. We have established a xenograft subcutaneous submillimeter tumor mouse model with HT-29-RFP by transplanting a single spheroid grown on radiation-crosslinked gelatin hydrogel microwells. Here, we developed an in vivo dual-observation method to observe the submillimeter tumor mass and tumor-surface blood vessels using this model. RFP was detected to observe the tumor mass, and a fluorescent angiography agent FITC-dextran was administered to observe blood vessels via stereoscopic fluorescence microscopy. The anti-angiogenesis agent regorafenib was used to confirm the usefulness of this method. This method effectively detected the submillimeter tumor mass and tumor-surface blood vessels in vivo. Regorafenib treatment revealed tumor growth inhibition and angiogenesis downregulation with reduced vascular extremities, segments, and meshes. Further, we confirmed that tumor-surface blood vessel areas monitored using in vivo dual-observation correlated with intratumoral blood vessel areas observed via fluorescence microscopy with frozen sections. In conclusion, this method would be useful in developing anti-angiogenesis agents against submillimeter tumors.

Microfluidic devices, which miniaturize cell culture and chemical experiments from lab-scale to microchip dimensions, have gained significant attention in recent years. Extensive research has been conducted on microfluidic mixers, which facilitate the mixing and agitation of chemicals. The “Sidewall-Driven Micromixer” that we are currently developing employs a unique mechanism; it induces a swirling flow within the main chamber by vibrating the silicone wall situated between the main and driving chambers using pressure fluctuations. In an earlier study, we found that Sidewall-Driven Micromixers of a size suitable for small cells could indeed produce this swirling flow. Furthermore, we successfully established concentration gradients within each mixer. However, when attempting to upscale the mixer while maintaining conventional proportions to accommodate larger cell aggregates such as spheroids, the desired swirling flow was not achieved. To address this challenge, we made adjustments to the wall dimensions, aiming to amplify wall deformation and thereby enhance the mixer’s driving force. Concurrently, we modified the mixer’s shape to ensure that the increased wall deformation would not hinder the fluid flow. These alterations not only improved the mixer’s performance but also provided valuable insights for positioning the mixer’s neck channel, considering the extent of wall deformation.

Overcoming drug resistance and specifically targeting cancer stem cells (CSCs) are critical challenges in improving cancer therapy. Nowadays, the use of novel and native medicinal plants can provide new sources for further investigations for this purpose. The aim of this study was to assess the potential of S. bachtiarica, an endemic plant with diverse medicinal applications, in suppressing and targeting cancer and cancer stem cells in glioblastoma and breast cancer. The effect of S. bachtiarica on viability, migration, invasion, and clonogenic potential of MDAMB-231 and U87-MG cells was assessed in both two- and three-dimensional cell culture models. Additionally, we evaluated its effects on the self-renewal capacity of mammospheres. The experimental outcomes indicated that S. bachtiarica decreased the viability and growth rate of cells and spheroids by inducing apoptosis and inhibited colony formation, migration, and invasion of cells and spheroids. Additionally, colony and sphere-forming ability, as well as the expression of genes associated with EMT and stemness were reduced in mammospheres treated with S. bachtiarica. In conclusion, this study provided valuable insights into the anti-cancer effects of S. bachtiarica, particularly in relation to breast CSCs. Therefore, S. bachtiarica may be a potential adjuvant for the treatment of cancer.

Functionally enhanced mesenchymal stromal cells participate in the repair of intervertebral disc. This study aimed to assess the safety and tolerability of intradiscal administration of matrilin-3-primed adipose-derived stromal cell (ASC) spheroids with hyaluronic acid (HA) in patients with chronic discogenic low back pain (LBP). In this single-arm, open-label phase I clinical trial, eight patients with chronic discogenic LBP were observed over 6 months. Each patient underwent a one-time intradiscal injection of 1 mL of 6.0 × 106 cells/disc combined with HA under real-time fluoroscopic guidance. Safety and feasibility were gauged using Visual Analogue Scale (VAS) pain and Oswestry Disability Index (ODI) scores and magnetic resonance imaging. All participants remained in the trial, with no reported adverse events linked to the procedure or stem cells. A successful outcome-marked by a minimum 2-point improvement in the VAS pain score and a 10-point improvement in ODI score from the start were observed in six participants. Although the modified Pfirrmann grade remained consistent across all participants, radiological improvements were evident in four patients. Specifically, two patients exhibited reduced high-intensity zones while another two demonstrated decreased disc protrusion. In conclusion, the intradiscal application of matrilin-3-primed ASC spheroids with HA is a safe and feasible treatment option for chronic discogenic LBP.

Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24–48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.

A basic process in cancer is the breaching of basement-membrane barriers to permit tissue invasion. Cancer cells can use proteases and physical mechanisms to produce initial holes in basement membranes, but how cells squeeze through this barrier into matrix environments is not well understood. We used a 3D invasion model consisting of cancer-cell spheroids encapsulated by a basement membrane and embedded in collagen to characterize the dynamic early steps in cancer-cell invasion across this barrier. We demonstrate that certain cancer cells extend exceptionally long (~30–100 μm) protrusions through basement membranes via actin and microtubule cytoskeletal function. These long protrusions use integrin adhesion and myosin II-based contractility to pull cells through the basement membrane for initial invasion. Concurrently, these long, organelle-rich protrusions pull surrounding collagen inward while propelling cancer cells outward through perforations in the basement-membrane barrier. These exceptionally long, contractile cellular protrusions can facilitate the breaching of the basement-membrane barrier as a first step in cancer metastasis.

A major challenge for future drug development comprises finding alternative models for drug screening. The use of animal models in research is highly controversial, with an ongoing debate on their ethical acceptability. Also, animal models are often poorly predictive of therapeutic outcomes due to the differences between animal and human physiological environments. In this study, we aimed to develop a biomimetic hydrogel that replicates the composition of skin for potential use in in vitro modeling within tissue engineering. The hydrogel was fabricated through the crosslinking of collagen type I, hyaluronic acid, four-arm PEG succinimidyl glutarate (4S-StarPEG), and fibrinogen. Various ratios of these components were systematically optimized to achieve a well-interconnected porosity and desirable rheological properties. To evaluate the hydrogel’s cytocompatibility, fibroblasts were embedded within the matrix. The resulting hydrogel exhibited promising properties as a scaffold, also facilitating the growth of and proliferation of the cells. This biomimetic hydrogel holds great potential for tissue engineering applications, particularly in skin regeneration and cancer research. The study used melanoma spheroids fabricated using the 96-round bottom well plate method as a potential application. The results demonstrate that the developed hydrogels allowed the maintenance of spheroid integrity and viability, meaning it has a promising use as a three-dimensional in vitro model of melanoma for both tissue engineering and drug screening applications.

Prior to clinical trials, preclinical testing of oncology drug candidates is performed by evaluating drug candidates with in vitro and in vivo platforms. For in vivo testing, animal models are used to evaluate the toxicity and efficacy of drug candidates. However, animal models often display poor translational results as many drugs that pass preclinical testing fail when tested with humans, with oncology drugs exhibiting especially poor acceptance rates. The FDA Modernization Act 2.0 promotes alternative preclinical testing techniques, presenting the opportunity to use higher complexity in vitro models as an alternative to in vivo testing, including three-dimensional (3D) cell culture models. Three-dimensional tissue cultures address many of the shortcomings of 2D cultures by more closely replicating the tumour microenvironment through a combination of physiologically relevant drug diffusion, paracrine signalling, cellular phenotype, and vascularization that can better mimic native human tissue. This review will discuss the common forms of 3D cell culture, including cell spheroids, organoids, organs-on-a-chip, and 3D bioprinted tissues. Their advantages and limitations will be presented, aiming to discuss the use of these 3D models to accurately represent human tissue and as an alternative to animal testing. The use of 3D culture platforms for preclinical drug development is expected to accelerate as these platforms continue to improve in complexity, reliability, and translational predictivity.

Keloid refers to a fibro-proliferative disorder characterized by an accumulation of extracellular matrix at the dermis level, overgrowing beyond the initial wound and forming tumor-like nodule areas. The absence of treatment for keloid is clearly related to limited knowledge about keloid etiology. In vitro, keloids were classically studied through fibroblasts monolayer culture, far from keloid in vivo complexity. Today, cell aggregates cultured as 3D spheroid have gained in popularity as new tools to mimic tissue in vitro. However, no previously published works on spheroids have specifically focused on keloids yet. Thus, we hypothesized that spheroids made of keloid fibroblasts (KFs) could be used to model fibrogenesis in vitro. Our objective was to qualify spheroids made from KFs and cultured in a basal or pro-fibrotic environment (+TGF-β1). As major parameters for fibrogenesis assessment, we evaluated apoptosis, myofibroblast differentiation and response to TGF-β1, extracellular matrix (ECM) synthesis, and ECM-related genes regulation in KFs spheroids. We surprisingly observed that fibrogenic features of KFs are strongly downregulated when cells are cultured in 3D. In conclusion, we believe that spheroid is not the most appropriate model to address fibrogenesis in keloid, but it constitutes an efficient model to study the deactivation of fibrotic cells.

Prediction of therapeutic outcomes is important for cancer patients in order to reduce side effects and improve the efficacy of anti-cancer drugs. Currently, the most widely accepted method for predicting the efficacy of anti-cancer drugs is gene panel testing based on next-generation sequencing. However, gene panel testing has several limitations. For example, only 10% of cancer patients are estimated to have druggable mutations, even if whole-exome sequencing is applied. Additionally, even if optimal drugs are selected, a significant proportion of patients derive no benefit from the indicated drug treatment. Furthermore, most of the anti-cancer drugs selected by gene panel testing are molecularly targeted drugs, and the efficacies of cytotoxic drugs remain difficult to predict. Apart from gene panel testing, attempts to predict chemotherapeutic efficacy using ex vivo cultures from cancer patients have been increasing. Several groups have retrospectively demonstrated correlations between ex vivo drug sensitivity and clinical outcome. For ex vivo culture, surgically resected tumor tissue is the most abundant source. However, patients with recurrent or metastatic tumors do not usually undergo surgery, and chemotherapy may be the only option for those with inoperable tumors. Therefore, predictive methods using small amounts of cancer tissue from diagnostic materials such as endoscopic, fine-needle aspirates, needle cores and liquid biopsies are needed. To achieve this, various types of ex vivo culture and endpoint assays using effective surrogate biomarkers of drug sensitivity have recently been developed. Here, we review the variety of ex vivo cultures and endpoint assays currently available.

Long-term administration of aspirin (ASA, acetylsalicylic acid) in oncogenic patients has been related to a reduction in cancer risk incidence, but its precise mechanism of action is unclear. The activation of cancer-associated fibroblasts (CAFs) is a key element in tumor progression and can be triggered by cancer-derived extracellular vesicles (EVs). Targeting the communication between cancer cells and the surrounding tumor microenvironment (TME) may control cancer progression. Our aim was to investigate the effect of ASA on breast cancer cells, focusing on EV secretion and their effect on the biological properties of CAFs. As a result, ASA was shown to reduce the amount and alter the size distribution of EVs produced by MDA-MB-231 tumor cells. Fibroblasts stimulated with EVs derived from MDA-MB-231 treated with ASA (EV-ASA) showed a lower expression of alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP2) but not fibroblast activation protein (FAP) in respect to the ones stimulated with EVs from untreated breast cancer cells (EV-CTR). Furthermore, invasion assays using a three-dimensional (3D) fibroblast spheroid model showed reduced MDA-MB-231 invasion towards fibroblast spheroids pretreated with EV-ASA as compared to spheroids prepared with EV-CTR-stimulated fibroblasts. This suggests that ASA partially inhibits the ability of tumor EVs to stimulate CAFs to promote cancer invasion. In conclusion, ASA can interfere with tumor communication by reducing EV secretion by breast tumor cells as well as by interfering with their capacity to stimulate fibroblasts to become CAFs.

Cancer is a multifaceted health issue that affects people globally and it is considered one of the leading causes of death with a high percentage of victims worldwide. In recent years, research studies have uncovered great advances in cancer diagnosis and treatment. But, there are still major drawbacks of the conventional therapies used including severe side effects, toxicity, and drug resistance. That is why it is critical to develop new drugs with advantages like low cytotoxicity and no treatment resistance to the cancer cells. Antimicrobial peptides (AMPs) have recently attracted attention as a novel therapeutic strategy for the treatment of various cancers, targeting tumor cells with less toxicity to normal tissues. The aim of the study was to discover alternate treatments that do not lead to cancer resistance and have fewer side effects. Here, we report the effects induced by several AMPs, Melittin, Cecropin A, and a Cecropin A—Melittin hybrid, against two human colorectal cancer-derived spheroids. To study the effects of the peptides, cell viability was investigated using MTT, LDH, and ATP assays. Furthermore, cellular senescence and cell cycle were investigated. We found that using different concentrations of these peptides affected the spheroids, their structure being highly compromised by reducing cell viability, and the increase in ATP and LDH levels. Also, the cells are arrested in the G2/M phase leading to an increase in senescent cells. We show that Melittin and the hybrid are most effective against the 3D colorectal cancer cells compared to Cecropin A.

An organoid is a 3D organization of cells that can recapitulate some of the structure and function of native tissue. Recent work has seen organoids gain prominence as a valuable model for studying tissue development, drug discovery, and potential clinical applications. The requirements for the successful culture of organoids in vitro differ significantly from those of traditional monolayer cell cultures. The generation and maturation of high-fidelity organoids entails developing and optimizing environmental conditions to provide the optimal cues for growth and 3D maturation, such as oxygenation, mechanical and fluidic activation, nutrition gradients, etc. To this end, we discuss the four main categories of bioreactors used for organoid culture: stirred bioreactors (SBR), microfluidic bioreactors (MFB), rotating wall vessels (RWV), and electrically stimulating (ES) bioreactors. We aim to lay out the state-of-the-art of both commercial and in-house developed bioreactor systems, their benefits to the culture of organoids derived from various cells and tissues, and the limitations of bioreactor technology, including sterilization, accessibility, and suitability and ease of use for long-term culture. Finally, we discuss future directions for improvements to existing bioreactor technology and how they may be used to enhance organoid culture for specific applications.

Breast cancer (BC) is a heterogenous disease classified into four molecular subtypes (Luminal A, Luminal B, HER2 and triple-negative (TNBC)) depending on the expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal receptor 2 (HER2). The development of effective treatments for BC, especially TNBC, remains a challenge. Aminosteroid derivative RM-581 has previously shown an antiproliferative effect in multiple cancers in vitro and in vivo. In this study, we evaluated its effect in BC cell lines representative of BC molecular subtypes, including metastatic TNBC. We found that RM-581 has an antiproliferative effect on all BC molecular subtypes, especially on Luminal A and TNBC, in 2D and 3D cultures. The combination of RM-581 and trastuzumab or trastuzumab-emtansine enhanced the anticancer effect of each drug for HER2-positive BC cell lines, and the combination of RM-581 and taxanes (docetaxel or paclitaxel) improved the antiproliferative effect of RM-581 in TNBC and metastatic TNBC cell lines. We also confirmed that RM-581 is an endoplasmic reticulum (EnR)-stress aggravator by inducing an increase in EnR-stress-induced apoptosis markers such as BIP/GRP78 and CHOP and disrupting lipid homeostasis. This study demonstrates that RM-581 could be effective for the treatment of BC, especially TNBC.

In order to improve the conversion and transmission efficiency of the photoelectron, core–shell spheroid structure titanium dioxide/cadmium sulfide (TiO2/CdS) composites were synthesized as epoxy-based coating fillers using a simple hydrothermal method. The electrochemical performance of photocathodic protection for the epoxy-based composite coating was analyzed by coating it on the Q235 carbon steel surface. The results show that the epoxy-based composite coating possesses a significant photoelectrochemical property with a photocurrent density of 0.0421 A/cm2 and corrosion potential of −0.724 V. Importantly, the modified composite coating can extend absorption in the visible region and effectively separate photoelectron hole pairs to improve the photoelectrochemical performance synergistically, because CdS can be regarded as a sensitizer to be introduced into TiO2 to form a heterojunction system. The mechanism of photocathodic protection is attributed to the potential energy difference between Fermi energy and excitation level, which leads to the system obtaining higher electric field strength at the heterostructure interface, thus driving electrons directly into the surface of Q235 carbon steel (Q235 CS). Moreover, the photocathodic protection mechanism of the epoxy-based composite coating for Q235 CS is investigated in this paper.

Cholangiocarcinoma (CCA) are characterized by their desmoplastic and hypervascularized tumor microenvironment (TME), which is mainly composed of tumor cells and cancer-associated fibroblasts (CAFs). CAFs play a pivotal role in general and CCA tumor progression, angiogenesis, metastasis, and the development of treatment resistance. To our knowledge, no continuous human in vivo-like co-culture model is available for research. Therefore, we aimed to establish a new model system (called MUG CCArly) that mimics the desmoplastic microenvironment typically seen in CCA. Proteomic data comparing the new CCA tumor cell line with our co-culture tumor model (CCTM) indicated a higher gene expression correlation of the CCTM with physiological CCA characteristics. A pro-angiogenic TME that is typically observed in CCA could also be better simulated in the CCTM group. Further analysis of secreted proteins revealed CAFs to be the main source of these angiogenic factors. Our CCTM MUG CCArly represents a new, reproducible, and easy-to-handle 3D CCA model for preclinical studies focusing on CCA-stromal crosstalk, tumor angiogenesis, and invasion, as well as the immunosuppressive microenvironment and the involvement of CAFs in the way that drug resistance develops.

Here we aimed to establish a simple detection method for detecting circulating tumor cells (CTCs) in the blood sample of colorectal cancer (CRC) patients using poly(2-methoxyethyl acrylate) (PMEA)-coated plates. Adhesion test and spike test using CRC cell lines assured efficacy of PMEA coating. A total of 41 patients with pathological stage II–IV CRC were enrolled between January 2018 and September 2022. Blood samples were concentrated by centrifugation by the OncoQuick tube, and then incubated overnight on PMEA-coated chamber slides. The next day, cell culture and immunocytochemistry with anti-EpCAM antibody were performed. Adhesion tests revealed good attachment of CRCs to PMEA-coated plates. Spike tests indicated that ~75% of CRCs from a 10-mL blood sample were recovered on the slides. By cytological examination, CTCs were identified in 18/41 CRC cases (43.9%). In cell cultures, spheroid-like structures or tumor-cell clusters were found in 18/33 tested cases (54.5%). Overall, CTCs and/or growing circulating tumor cells were found in 23/41 CRC cases (56.0%). History of chemotherapy or radiation was significantly negatively correlated with CTC detection (p = 0.02). In summary, we successfully captured CTCs from CRC patients using the unique biomaterial PMEA. Cultured tumor cells will provide important and timely information regarding the molecular basis of CTCs.

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents.

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be crucial for tumor angiogenesis, invasion, and metastasis, and thus MT1-MMP is a high priority target for potential cancer therapies. To properly evaluate MT1-MMP inhibitors, a screening protocol is desired by which enzyme activity can be quantified in a tumor microenvironment-like model system. In the present study, we applied a fluorogenic, collagen model triple-helical substrate to quantify MT1-MMP activity for tumor spheroids embedded in a collagen hydrogel. The substrate was designed to be MT1-MMP selective and to possess fluorescent properties compatible with cell-based assays. The proteolysis of the substrate correlated to glioma spheroid invasion. In turn, the application of either small molecule or protein-based MMP inhibitors reduced proteolytic activity and glioma spheroid invasion. The presence of MT1-MMP in glioma spheroids was confirmed by western blotting. Thus, spheroid invasion was dependent on MT1-MMP activity, and inhibitors of MT1-MMP and invasion could be conveniently screened in a high-throughput format. The combination of the fluorogenic, triple-helical substrate, the three-dimensional tumor spheroids embedded in collagen, and Hit-Pick software resulted in an easily adaptable in vivo-like tumor microenvironment for rapidly processing inhibitor potential for anti-cancer use.

Tissue organoids hold enormous potential as tools for a variety of applications, including disease modeling and drug screening. To effectively mimic the native tissue environment, it is critical to integrate a microvasculature with the parenchyma and stroma. In addition to providing a means to physiologically perfuse the organoids, the microvasculature also contributes to the cellular dynamics of the tissue model via the cells of the perivascular niche, thereby further modulating tissue function. In this review, we discuss current and developing strategies for vascularizing organoids, consider tissue-specific vascularization approaches, discuss the importance of perfusion, and provide perspectives on the state of the field.

Spheroids are expected to aid the establishment of an in vitro-based cell culture system that can realistically reproduce cellular dynamics in vivo. We developed a fluoropolymer scaffold with an extracellular matrix (ECM) dot array and confirmed the possibility of mass-producing spheroids with uniform dimensions. Controlling the quality of ECM dots is important as it ensures spheroid uniformity, but issues such as pattern deviation and ECM drying persist in the conventional microstamping method. In this study, these problems were overcome via ECM dot printing using a resin mask with dot-patterned holes. For dot diameters of φ 300 μm, 400 μm, and 600 μm, the average spheroid diameters of human iPS cells (hiPSCs) were φ 260.8 μm, 292.4 μm, and 330.7 μm, respectively. The standard deviation when each average was normalized to 100 was 14.1%. A high throughput of 89.9% for colony formation rate to the number of dots and 89.3% for spheroid collection rate was achieved. The cells proliferated on ECM dots, and the colonies could be naturally detached from the scaffold without the use of enzymes, so there was almost no stimulation of the cells. Thus, the undifferentiated nature of hiPSCs was maintained until day 4. Therefore, this method is expected to be useful in drug discovery and regenerative medicine.

Introduction: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. Results: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. Conclusion: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state.

A 3D tumor spheroid has been increasingly applied in pharmaceutical development for its simulation of the tumor structure and microenvironment. The embedded-culture of a tumor spheroid within a hydrogel microenvironment could help to improve the mimicking of in vivo cell growth and the development of 3D models for tumor invasiveness evaluation, which could enhance its drug efficiency prediction together with cell viability detection. NCI-H23 spheroids and CT-26 spheroids, from a non–small cell lung cancer and colorectal cancer cell line, respectively, together with extracellular matrix were generated for evaluating their sensitivity to AMG510 (a KRASG12C inhibitor) under normoxia and hypoxia conditions, which were created by an on-stage environmental chamber. Results demonstrated that NCI-H23, the KRASG12C moderate expression cell line, only mildly responded to AMG510 treatment in normal 2D and 3D cultures and could be clearly evaluated by our system in hypoxia conditions, while the negative control CT-26 (G12D-mutant) spheroid exhibited no significant response to AMG510 treatment. In summary, our system, together with a controlled microenvironment and imaging methodology, provided an easily assessable and effective methodology for 3D in vitro drug efficiency testing and screenings.

The liver produces and stores various nutrients that are necessary for the body and serves as a chemical plant, metabolizing carbohydrates, fats, hormones, vitamins, and minerals. It is also a vital organ for detoxifying drugs and exogenous harmful substances. Culturing liver cells in vitro under three-dimensional (3D) conditions is considered a primary mechanism for liver tissue engineering. The 3D cell culture system is designed to allow cells to interact in an artificially created environment and has the advantage of mimicking the physiological characteristics of cells in vivo. This system facilitates contact between the cells and the extracellular matrix. Several technically different approaches have been proposed, including bioreactors, chips, and plate-based systems in fluid or static media composed of chemically diverse materials. Compared to conventional two-dimensional monolayer culture in vitro models, the ability to predict the function of the tissues, including the drug metabolism and chemical toxicity, has been enhanced by developing three-dimensional liver culture models. This review discussed the methodology of 3D cell cultures and summarized the advantages of an in vitro liver platform using 3D culture technology.

3-D cell cultures are being increasingly used as in vitro models are capable of better mimicry of in vivo tissues, particularly in drug screenings where mass transfer limitations can affect the cancer biology and response to drugs. Three-dimensional microscopy techniques, such as confocal and multiphoton microscopy, have been used to elucidate data from 3-D cell cultures and whole organs, but their reach inside the 3-D tissues is restrained by the light scattering of the tissues, limiting their effective reach to 100–200 µm, which is simply not enough. Tissue clearing protocols, developed mostly for larger specimens usually involve multiple steps of viscous liquid submersion, and are not easily adaptable for much smaller spheroids and organoids. In this work, we have developed a novel tissue clearing solution tailored for small spheroids and organoids. Our tissue clearing protocol, called HyClear, uses a mixture of DMSO, HPG and urea to allow for one-step tissue clearing of spheroids and organoids, and is compatible with high-throughput screening studies due to its speed and simplicity. We have shown that our tissue clearing agent is superior to many of the commonly used tissue clearing agents and allows for elucidating better quality data from drug screening experiments.

Cancer cells may stimulate glycolytic flux when O2 becomes insufficient. Increase in L-lactate release therefore appears as an escape mechanism to drugs targeting mitochondrial respiration but also represents a response that may be exploited to screen for compounds blocking either mitochondrial carriers of oxidizable substrates or the electron transport chain. Here, we developed a screening procedure based on the capacity of cancer cells to release L-lactate to gain insights on the development of mitochondrial complex I inhibitors. For this purpose, we synthesized derivatives of carboxyamidotriazole, a compound previously described as a potential OXPHOS inhibitor. Two series of derivatives were generated by cycloaddition between benzylazide and either cyanoacetamides or alkynes. A primary assay measuring L-lactate release as a compensatory mechanism upon OXPHOS inhibition led us to identify 15 hits among 28 derivatives. A secondary assay measuring O2 consumption in permeabilized cancer cells confirmed that 12 compounds among the hits exhibited reversible complex I inhibitory activity. Anticancer effects of a short list of 5 compounds identified to induce more L-lactate release than reference compound were then evaluated on cancer cells and tumor-mimicking 3D spheroids. Human and mouse cancer cell monolayers exhibiting high level of respiration in basal conditions were up to 3-fold more sensitive than less oxidative cancer cells. 3D tumor spheroids further revealed potency differences between selected compounds in terms of cytotoxicity but also radiosensitizing activity resulting from local reoxygenation. In conclusion, this study documents the feasibility to efficiently screen in 96-well plate format for mitochondrial complex I inhibitors based on the capacity of drug candidates to induce L-lactate release.

Photon-based radiotherapy upregulates Notch signaling in cancer, leading to the acquisition of the stem cell phenotype and induction of invasion/migration, which contributes to the development of resistance to therapy. However, the effect of carbon ion radiotherapy (CIRT) on Notch signaling in glioma and its impact on stemness and migration is not explored yet. Human glioma cell lines (LN229 and U251), stable Notch1 intracellular domain (N1ICD) overexpressing phenotype of LN229 cells, and Notch inhibitor resistant LN229 cells (LN229R) were irradiated with either photon (X-rays) or (carbon ion irradiation) CII, and expressions of Notch signaling components were accessed by RT-PCR, Western blotting, and enzymatic assays and flow cytometry. Spheroid forming ability, cell migration, and clonogenic assay were used to evaluate the effect of modulated Notch signaling by irradiation. Our results show that X-ray irradiation induced the expression of Notch signaling components such as Notch receptors, target genes, and ADAM17 activity, while CII reduced it in glioma cell lines. The differential modulation of ADAM17 activity by CII and X-rays affected the cell surface levels of NOTCH1 and NOTCH2 receptors, as they were reduced by X-ray irradiation but increased in response to CII. Functionally, CII reduced the spheroid formation and migration of glioma cells, possibly by downregulating the N1ICD, as stable overexpression of N1ICD rescued these inhibitory effects of CII. Moreover, LN229R that are less reliant on Notch signaling for their survival showed less response to CII. Therefore, downregulation of Notch signaling resulting in the suppression of stemness and impaired cell migration by CII seen here may reduce tumor regrowth and disease dissemination, in addition to the well-established cytotoxic effects.

Cell-based assays, conducted on monolayer (2D) cultured cells, are an unquestionably valuable tool for biomedical research. However, three-dimensional (3D) cell culture models have gained relevance over the last few years due to the advantages of better mimicking the microenvironment and tissue microarchitecture in vivo. Recent magnetic-based 3D (m3D) cell culture systems can be used for this purpose. These systems are based on exposing magnetized cells to magnetic fields by levitation, bioprinting, or ring formation to promote cell aggregation into 3D structures. However, the successful development of these structures is dependent on several methodological characteristics and can be applied to mimic different human tissues. Thus, a systematic review was performed using Medline (via Pubmed), Scopus, and Web of Science (until February 2022) databases to aggregate studies using m3D culture in which human tissues were mimicked. The search generated 3784 records, of which 25 met the inclusion criteria. The usability of these m3D systems for the development of homotypic or heterotypic spheroids with or without scaffolds was explored in these studies. We also explore methodological differences specifically related to the magnetic method. Generally, the development of m3D cultures has been increasing, with bioprinting and levitation systems being the most used to generate homotypic or heterotypic cultures, mainly to mimic the physiology of human tissues, but also to perform therapeutic screening. This systematic review showed that there are areas of research where the application of this method remains barely explored, such as cancer research.

Up-to-date in vitro and in vivo preclinical models expressing the patient-specific cancer lineage responsible for CRC and its metastatic behavior and responsiveness to therapy are needed. Exosomes’ role in tumorigenesis and the metastatic process was demonstrated, and the material content and size of the exosomes are associated with a poor prognosis of CRC. Exosomes are generally imagined after their recovery from blood serum as isolated entities, and our work aims to investigate them “in situ” in their native environment by scanning and transmission electron microscopy to understand their secretion modalities. We studied CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and in their mouse xenograft to find possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. We observed that MTSs’ exosome secretion patterns depend on their structural complexity: few-layer MTSs show a lesser exosome secretion, limited to the apical domain of cancer cells, secretion increases in multilayered MTSs, and it develops from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in MTSs. This difference in exosome secretion pattern between MTSs and xenografts may be due to the influence of surrounding non-tumor cells.

The 3Rs guidelines recommend replacing animal testing with alternative models. One of the solutions proposed is organ-on-chip technology in which liver-on-chip is one of the most promising alternatives for drug screening and toxicological assays. The main challenge is to achieve the relevant in vivo-like functionalities of the liver tissue in an optimized cellular microenvironment. Here, we investigated the development of hepatic cells under dynamic conditions inside a 3D hydroscaffold embedded in a microfluidic device. The hydroscaffold is made of hyaluronic acid and composed of liver extracellular matrix components (galactosamine, collagen I/IV) with RGDS (Arg-Gly-Asp-Ser) sites for cell adhesion. The HepG2/C3A cell line was cultured under a flow rate of 10 µL/min for 21 days. After seeding, the cells formed aggregates and proliferated, forming 3D spheroids. The cell viability, functionality, and spheroid integrity were investigated and compared to static cultures. The results showed a 3D aggregate organization of the cells up to large spheroid formations, high viability and albumin production, and an enhancement of HepG2 cell functionalities. Overall, these results highlighted the role of the liver-on-chip model coupled with a hydroscaffold in the enhancement of cell functions and its potential for engineering a relevant liver model for drug screening and disease study.

Aspects related to the response of cells to photodynamic therapy (PDT) have been well studied in cell cultures, which often grow in monolayers. In this work, we propose a spheroidal model of U87MG and SKBR3 cells designed to mimic superficial tumor tissue, small spheroids (500 µm) that can be xenografted onto quail chorioallantoic membrane (CAM) to study the effects of PDT in real time. Hypericin was used as a model molecule for a hydrophobic photosensitizer that can produce singlet oxygen (1O2). 1O2 production by hypericin was detected in SKBR3 and U87MG spheroid models using a label-free technique. Vital fluorescence microscopy and flow cytometry revealed the heterogeneity of caspase-3 distribution in the cells of the spheroids. The levels of caspase-3 and apoptosis increased in the cells of spheroids 24 h after PDT. Lactate dehydrogenase activity was evaluated in the spheroids as the most reliable assay to detect differences in phototoxicity. Finally, we demonstrated the applicability of U87MG spheroids on CAM in photodiagnostics. Overall, the variability and applicability of the prepared spheroid models were demonstrated in the PDT study.

The blood–brain barrier (BBB) is an essential structure for the maintenance of brain homeostasis. Alterations to the BBB are linked with a myriad of pathological conditions and play a significant role in the onset and evolution of neurodegenerative diseases, including Alzheimer’s disease. Thus, a deeper understanding of the BBB’s structure and function is mandatory for a better knowledge of neurodegenerative disorders and the development of effective therapies. Because studying the BBB in vivo imposes overwhelming difficulties, the in vitro approach remains the main possible way of research. With many in vitro BBB models having been developed over the last years, the main aim of this review is to systematically present the most relevant designs used in neurological research. In the first part of the article, the physiological and structural–functional parameters of the human BBB are detailed. Subsequently, available BBB models are presented in a comparative approach, highlighting their advantages and limitations. Finally, the new perspectives related to the study of Alzheimer’s disease with the help of novel devices that mimic the in vivo human BBB milieu gives the paper significant originality.

Compared with traditional two-dimensional culture, a three-dimensional (3D) culture platform can not only provide more reliable prediction results, but also provide a simple, inexpensive and less time-consuming method compared with animal models. A direct in vitro model of the patient’s tumor can help to achieve individualized and precise treatment. However, the existing 3D culture system based on microwell arrays has disadvantages, such as poor controllability, an uneven spheroid size, a long spheroid formation time, low-throughput and complicated operation, resulting in the need for considerable labor, etc. Here, we developed a new type of microdevice based on a 384-well plate/96-well plate microarray design. With our design, cells can quickly aggregate into clusters to form cell spheroids with better roundness. This design has the advantage of high throughput; the throughput is 33 times that of a 384-well plate. This novel microdevice is simple to process and convenient to detect without transferring the cell spheroid. The results show that the new microdevice can aggregate cells into spheroids within 24 h and can support drug and radiation sensitivity analyses in situ in approximately one week. In summary, our microdevices are fast, efficient, high-throughput, simple to process and easy to detect, providing a feasible tool for the clinical validation of individualized drug/radiation responses in patients.

Nanoconstructs developed for biomedical purposes must overcome diverse biological barriers before reaching the target where playing their therapeutic or diagnostic function. In vivo models are very complex and unsuitable to distinguish the roles plaid by the multiple biological barriers on nanoparticle biodistribution and effect; in addition, they are costly, time-consuming and subject to strict ethical regulation. For these reasons, simplified in vitro models are preferred, at least for the earlier phases of the nanoconstruct development. Many in vitro models have therefore been set up. Each model has its own pros and cons: conventional 2D cell cultures are simple and cost-effective, but the information remains limited to single cells; cell monolayers allow the formation of cell–cell junctions and the assessment of nanoparticle translocation across structured barriers but they lack three-dimensionality; 3D cell culture systems are more appropriate to test in vitro nanoparticle biodistribution but they are static; finally, bioreactors and microfluidic devices can mimicking the physiological flow occurring in vivo thus providing in vitro biological barrier models suitable to reliably assess nanoparticles relocation. In this evolving context, the present review provides an overview of the most representative and performing in vitro models of biological barriers set up for nanomedical research.

Ovarian cancer (OC) is one of the most common gynecological cancers, with the worst prognosis and the highest mortality rate. Peritoneal dissemination (or carcinomatosis) accompanied by ascites formation is the most unfavorable factor in the progression and recurrence of OC. Tumor cells in ascites are present as either separate cells or, more often, as cell aggregates, i.e., spheroids which promote implantation on the surface of nearby organs and, at later stages, metastases to distant organs. Malignant ascites comprises a unique tumor microenvironment; this fact may be of relevance in the search for new prognostic and predictive factors that would make it possible to personalize the treatment of patients with OC. However, the precise mechanisms of spheroid formation and carcinomatosis are still under investigation. Here, we summarize data on ascites composition as well as the activity of fibroblasts and macrophages, the key stromal and immune components, in OC ascites. We describe current knowledge about the role of fibroblasts and macrophages in tumor spheroid formation, and discuss the specific functions of fibroblasts, macrophages and T cells in tumor peritoneal dissemination and implantation.

In this study, we proposed an in vitro tumor model to simulate the mechanical microenvironment and investigate the effect of compressive stress on the invasion process of malignant tumors. It has been pointed out that the biomechanical environment, as well as the biochemical environment, could affect the transformation of cancer cell migration, invasion, and metastasis. We hypothesized that the solid stress caused by the exclusion of surrounding tissue could transform tumor cells from noninvasive to invasive phenotypes. Colorectal cell spheroids were embedded and cultured in agarose gels of varying concentrations to simulate the earliest stages of tumor formation and invasion. The spheroids embedded in gels at higher concentrations showed peculiar growth after 72 h of culture, and the external compressive loading imposed on them caused peculiar growth even in the gels at lower concentrations. In conclusion, the mechanical microenvironment caused the transformation of tumor cell phenotypes, promoting the growth and invasion of tumor cell spheroids.

Cartilage is a connective tissue that constitutes the structure of the body and consists of chondrocytes that produce considerable collagenous extracellular matrix and plentiful ground substances, such as proteoglycan and elastin fibers. Self-repair is difficult when the cartilage is damaged because of insufficient blood supply, low cellularity, and limited progenitor cell numbers. Therefore, three-dimensional (3D) culture systems, including pellet culture, hanging droplets, liquid overlays, self-injury, and spinner culture, have attracted attention. In particular, 3D spheroid culture strategies can enhance the yield of exosome production of mesenchymal stem cells (MSCs) when compared to two-dimensional culture, and can improve cellular restorative function by enhancing the paracrine effects of MSCs. Exosomes are membrane-bound extracellular vesicles, which are intercellular communication systems that carry RNAs and proteins. Information transfer affects the phenotype of recipient cells. MSC-derived exosomes can facilitate cartilage repair by promoting chondrogenic differentiation and proliferation. In this article, we reviewed recent major advances in the application of 3D culture techniques, cartilage regeneration with stem cells using 3D spheroid culture system, the effect of exosomes on chondrogenic differentiation, and chondrogenic-specific markers related to stem cell derived exosomes. Furthermore, the utilization of MSC-derived exosomes to enhance chondrogenic differentiation for osteoarthritis is discussed. If more mechanistic studies at the molecular level are conducted, MSC-spheroid-derived exosomes will supply a better therapeutic option to improve osteoarthritis.

In this paper we present an efficient Matlab computation of a 3-D electromagnetic scattering problem, in which a plane wave impinges with a generic inclination onto a conducting ellipsoid of revolution. This solid is obtained by the rotation of an ellipse around one of its axes, which is also known as a spheroid. We have developed a fast and ad hoc code to solve the electromagnetic scattering problem, using spheroidal vector wave functions, which are special functions used to describe physical problems in which a prolate or oblate spheroidal reference system is considered. Numerical results are presented, both for TE and TM polarization of the incident wave, and are validated by a comparison with results obtained by a commercial electromagnetic simulator.

Background: In vitro three-dimensional (3D) hepatic spheroid culture has shown great promise in toxicity testing because it better mimics the cell–cell and cell–matrix interactions found in in vivo conditions than that of the traditional two-dimensional (2D) culture. Despite embedding HepaRG spheroids with collagen type I (collagen I) extracellular matrix (ECM) revealed a much better differentiation capability, almost all the collagen utilized in in vitro hepatocytes cultures is animal-derived collagen that may limit its use in human toxicity testing. Method: Here, a preliminary investigation of HepaRG cells cultured in different dimensionalities and with the addition of ECM was performed. Comparisons of conventional 2D culture with 3D spheroid culture were performed based on their functional or structural differences over 7 days. Rat tail collagen (rtCollagen) I and recombinant human collagen (rhCollagen) I were investigated for their ability in promoting HepaRG spheroid differentiation. Results: An immunofluorescence analysis of the hepatocyte-specific functional protein albumin suggested that HepaRG spheroids demonstrated better hepatic function than spheroids from 2D culture, and the function of HepaRG spheroids improved in a time-dependent manner. The fluorescence intensities per unit area of spheroids formed by 1000 cells on days 7 and 10 were 25.41 and 45.38, respectively, whereas almost undetectable fluorescence was obtained with 2D cells. In addition, the embedding of HepaRG spheroids into rtCollagen and rhCollagen I showed that HepaRG differentiation can be accelerated relative to the differentiation of spheroids grown in suspension, demonstrating the great promise of HepaRG spheroids. Conclusions: The culture conditions established in this study provide a potentially novel alternative for promoting the differentiation of HepaRG spheroids into mature hepatocytes through a collagen-embedded in vitro liver spheroid model. This culture method is envisioned to provide insights for future drug toxicology.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and has a unique metastatic route using ascites, known as the transcoelomic root. However, studies on ascites and contained cellular components have not yet been sufficiently clarified. In this review, we focus on the significance of accumulating ascites, contained EOC cells in the form of spheroids, and interaction with non-malignant host cells. To become resistant against anoikis, EOC cells form spheroids in ascites, where epithelial-to-mesenchymal transition stimulated by transforming growth factor-β can be a key pathway. As spheroids form, EOC cells are also gaining the ability to attach and invade the peritoneum to induce intraperitoneal metastasis, as well as resistance to conventional chemotherapy. Recently, accumulating evidence suggests that EOC spheroids in ascites are composed of not only cancer cells, but also non-malignant cells existing with higher abundance than EOC cells in ascites, including macrophages, mesothelial cells, and lymphocytes. Moreover, hetero-cellular spheroids are demonstrated to form more aggregated spheroids and have higher adhesion ability for the mesothelial layer. To improve the poor prognosis, we need to elucidate the mechanisms of spheroid formation and interactions with non-malignant cells in ascites that are a unique tumor microenvironment for EOC.

Spheroids are recognized for resembling the important characteristics of natural tumors in cancer research. However, the lack of controllability of the spheroid size, form, and density in conventional spheroid culture methods reduces the reproducibility and precision of bioassay results and the assessment of drug-dose responses in spheroids. Nonetheless, the accurate prediction of cellular responses to drug compounds is crucial for developing new efficient therapeutic agents and optimizing existing therapeutic strategies for personalized medicine. We developed a surface-optimized PDMS microfluidic biochip to produce uniform and homogenous multicellular spheroids in a reproducible manner. This platform is surface optimized with 10% bovine serum albumin (BSA) to provide cell-repellent properties. Therefore, weak cell-surface interactions lead to the promotion of cell self-aggregations and the production of compact and uniform spheroids. We used a lung cancer cell line (A549), a co-culture model of lung cancer cells (A549) with (primary human osteoblasts, and patient-derived spine metastases cells (BML, bone metastasis secondary to lung). We observed that the behavior of cells cultured in three-dimensional (3D) spheroids within this biochip platform more closely reflects in vivo-like cellular responses to a chemotherapeutic drug, Doxorubicin, rather than on 24-well plates (two-dimensional (2D) model). It was also observed that the co-culture and patient-derived spheroids exhibited resistance to anti-cancer drugs more than the mono-culture spheroids. The repeatability of drug test results in this optimized platform is the hallmark of the reproducibility of uniform spheroids on a chip. This surface-optimized biochip can be a reliable platform to generate homogenous and uniform spheroids to study and monitor the tumor microenvironment and for drug screening.

Three-dimensional cell culture techniques mimic the in vivo cell environment more adequately than flat surfaces. Spheroids are multicellular aggregates and we aimed to produce scaffold-free spheroids of myogenic origin, called myospheres, using a mid-scale incubator and bioreactor hybrid. For the first time, we obtained spheroids from primary porcine muscle cells (PMCs) with this technology and compared their morphology and growth parameters, marker expression, and myogenic potential to C2C12-derived spheroids. Both cell types were able to form round-shaped spheroids in the bioreactor already after 24 h. The mean diameter of the C2C12 spheroids (44.6 µm) was larger than that of the PMCs (32.7 µm), and the maximum diameter exceeded 1 mm. C2C12 cells formed less aggregates than PMCs with a higher packing density (cell nuclei/mm2). After dissociation from the spheroids, C2C12 cells and PMCs started to proliferate again and were able to differentiate into the myogenic lineage, as shown by myotube formation and the expression of F-Actin, Desmin, MyoG, and Myosin. For C2C12, multinucleated syncytia and Myosin expression were observed in spheroids, pointing to accelerated myogenic differentiation. In conclusion, the mid-scale incubator and bioreactor system is suitable for spheroid formation and cultivation from primary muscle cells while preserving their myogenic potential.

Focused ultrasound (FUS) is a non-invasive technique producing a variety of biological effects by either thermal or mechanical mechanisms of ultrasound interaction with the targeted tissue. FUS could bring benefits, e.g., tumour sensitisation, immune stimulation, and targeted drug delivery, but investigation of FUS effects at the cellular level is still missing. New techniques are commonly tested in vitro on two-dimensional (2D) monolayer cancer cell culture models. The 3D tumour model—spheroid—is mainly utilised to mimic solid tumours from an architectural standpoint. It is a promising method to simulate the characteristics of tumours in vitro and their various responses to therapeutic alternatives. This study aimed to evaluate the effects of FUS on human prostate and glioblastoma cancer tumour spheroids in vitro. The experimental follow-up enclosed the measurements of spheroid integrity and growth kinetics, DNA damage, and cellular metabolic activity by measuring intracellular ATP content in the spheroids. Our results showed that pulsed FUS treatment induced molecular effects in 3D tumour models. With the disruption of the spheroid integrity, we observed an increase in DNA double-strand breaks, leading to damage in the cancer cells depending on the cancer cell type.

Spheroids have become an essential tool in preclinical cancer research. The uniformity of spheroids is a critical parameter in drug test results. Spheroids form by self-assembly of cells. Hence, the control of homogeneity of spheroids in terms of size, shape, and density is challenging. We developed surface-optimized polydimethylsiloxane (PDMS) biochip platforms for uniform spheroid formation on-chip. These biochips were surface modified with 10% bovine serum albumin (BSA) to effectively suppress cell adhesion on the PDMS surface. These surface-optimized platforms facilitate cell self-aggregations to produce homogenous non-scaffold-based spheroids. We produced uniform spheroids on these biochips using six different established human cell lines and a co-culture model. Here, we observe that the concentration of the BSA is important in blocking cell adhesion to the PDMS surfaces. Biochips treated with 3% BSA demonstrated cell repellent properties similar to the bare PDMS surfaces. This work highlights the importance of surface modification on spheroid production on PDMS-based microfluidic devices.